SIK2 inhibition enhances PARP inhibitor activity synergistically in ovarian and triple-negative breast cancers

Poly(ADP-ribose) polymerase inhibitors (PARP inhibitors) have had an increasing role in the treatment of ovarian and breast cancers. PARP inhibitors are selectively active in cells with homologous recombination DNA repair deficiency caused by mutations in BRCA1/2 and other DNA repair pathway genes. Cancers with homologous recombination DNA repair proficiency respond poorly to PARP inhibitors. Cancers that initially respond to PARP inhibitors eventually develop drug resistance. We have identified salt-inducible kinase 2 (SIK2) inhibitors, ARN3236 and ARN3261, which decreased DNA double-strand break (DSB) repair functions and produced synthetic lethality with multiple PARP inhibitors in both homologous recombination DNA repair deficiency and proficiency cancer cells. SIK2 is required for centrosome splitting and PI3K activation and regulates cancer cell proliferation, metastasis, and sensitivity to chemotherapy. Here, we showed that SIK2 inhibitors sensitized ovarian and triple-negative breast cancer (TNBC) cells and xenografts to PARP inhibitors. SIK2 inhibitors decreased PARP enzyme activity and phosphorylation of class-IIa histone deacetylases (HDAC4/5/7). Furthermore, SIK2 inhibitors abolished class-IIa HDAC4/5/7–associated transcriptional activity of myocyte enhancer factor-2D (MEF2D), decreasing MEF2D binding to regulatory regions with high chromatin accessibility in FANCD2, EXO1, and XRCC4 genes, resulting in repression of their functions in the DNA DSB repair pathway. The combination of PARP inhibitors and SIK2 inhibitors provides a therapeutic strategy to enhance PARP inhibitor sensitivity for ovarian cancer and TNBC.

. Source and culture medium of cell lines  The map contains changes that are color coded with red corresponding to up-regulation and green to downregulation. (B) Analysis of DNA repair and apoptosis genes. Gene expression was analyzed using RT-qPCR in SKOv3 and OVCAR8 ovarian cancer cells. Cells were treated with ARN3236, ARN3261, olaparib alone or olaparib+ARN3236 or ARN3261 for 72 hrs. The concentrations of ARN3236, ARN3261 and olaparib are 4µM (2 times), 4µM (3 times), and 15µM (2 times), respectively. The columns indicate the mean of RNA expression and the bars indicate the S.D. One-way ANOVA Tukey's multiple comparisons were performed (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001). Representative experiments were from one independent experiment with 3 technical repeats per treatment. Experiments were repeated three times with similar results. (C) Quantification of DNA damage using comet assays. Cells were plated and treated with ARN3236, ARN3261 or olaparib on the comet slides for a total of 48 hrs with and without olaparib for 16 hrs before harvest. HCC5032, OVCAR8 and SKOv3 were incubated with 1 µM of ARN3236 and MDA-MB-231 cells with 0.5 µM. All 4 cell lines tested were treated with 4 µM of ARN3261 and 5 µM of olaparib. Olive Tail Moment was measured using CaspLab1.2.3β2 software.
The columns indicate the mean of tail moments and the bars indicate the S.D. One-way ANOVA Tukey's multiple comparisons were performed (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001). Representative data were from three independent experiments with a total of 100-200 cells per treatment. (D) Quantification of DNA damage (-H2AX). Endogenous -H2AX was stained with anti--H2AX antibody in the cells treated with single agent or combined for 24 hrs. The concentration of ARN3261 is 1 µM. -H2AX dots were quantified with OLYMPUS CellSens Dimension software. T-test (two-tails) was performed between treatment and control (ns p>0.05, *** p<0.001, **** p<0.0001). Experiments were from two independent experiments with a total of 100 cells per treatment. Figure S4. ARN3236 and ARN3261 decrease phosphorylation of HDAC4/5/7 and promoter activity of MEF2 transcription factors. (A) Detection of HDAC5 localization with and without SIK2 inhibitors using immunofluorescence staining. Nuclear fluorescence intensity was measured by ImageJ (related to Figure 7A). Data are from three independent experiments with a total of 100-150 cells per group. The middle solid lines indicate the mean of fluorescent intensity. The top and bottom solid lines indicate the S.D. One-way ANOVA Tukey's multiple comparisons analysis was performed (*** p<0.001, **** p<0.0001). (B) Detection of HDAC5 localization with and without SIK2 inhibitors using cell fractionation. OVCAR8 and MDA-MB-231 cells were treated with ARN3236 (6 µM) or ARN3261 (5 µM) for 26 hrs. Total cell lyses were collected for cell fractionation and cytoplasmic extracts and nuclear extracts were subjected to western analysis using the antibodies indicated.
(D and L indicate dark and light exposure, respectively). Experiments were repeated twice with similar outcomes.
(C) Quantification of MEF2 promoter activity (related to Figure 7B). Cells were plated and after overnight incubation, then transfected with a mixture of a MEF2-responsive luciferase construct and a constitutively expressing Renilla luciferase construct (40:1) (QIAGEN) for 24 hrs. Cells were re-plated into 96 well plates and then treated with olaparib (4 µM) for different time intervals or with different doses of olaparib for 24 hrs as indicated. Cells were lysed for dual luciferase assay. The relative luciferase activity of MEF2 was calculated by normalizing to Renilla luciferase activity. The columns indicate the mean of MEF2 luciferase activity, and the bars indicate the S.D. One-way ANOVA Tukey's multiple comparisons analysis was performed ( ns p>0.05, * p<0.05). Representative experiments were from one independent experiment with 3 technical repeats per treatment. Experiments were repeated twice with similar outcomes. (D) Quantification of MEF2 promoter activity (related to Figure 7C). Cells were treated with TMP195 for 24 hrs prior to transfection of a mixture of a MEF2responsive luciferase construct and Renilla luciferase construct. Cells were re-plated into 96 well plate and then treated with ARN3236 (4 µM) and ARN3261 (4 µM) for 24 hrs. Measurement of luciferase activity was performed, quantified and analyzed as described in (    NextSeq500 sequencer using the High-output 75 single read configuration. The raw reads were first preprocessed to remove sequencing adapters and low-quality reads. The trimmed reads were then mapped to human reference genome hg19 using bowtie (1), with only uniquely mapped reads retained. The ChIP-seq occupancy profiles were generated by MACS 1.4 (2) with the "--wig" parameter, and were normalized to 20 million total reads. Duplicated reads were automatically removed by MACS. ChIPseq peaks were called by MACS with p-value set to 1e-8. Peaks were annotated to associated genes according to their relative locations.
T associated genes were identified as ChIP-seq target genes. Further functional analysis on these genes were carried out, including gene ontology (GO) analysis using DAVID (3,4). The enriched DNA binding motifs in ChIPseq peak regions were identified and compared with known motifs using HOMER v4.8 (5). The cleaved RNA fragments were then reverse transcribed into first strand cDNA by reverse transcriptase using random hexamer primers for RT priming and reverse transcription, followed by second strand cDNA synthesis using DNA polymerase I and RNase H. These double strand cDNA fragments were end-repaired and then adenylated at 3' ends with the addition of a single 'A' base to prevent self-ligation during subsequent ligation to the Illumina index-specific adapters that has a single "T" at 3' end which provides complementary overhang for ligating the adapter to the fragment. The raw library products were purified and enriched by PCR to create the final cDNA sequencing library. The indexed individual sequencing library was quantified using an Agilent Bioanalyzer Highly Sensitive DNA assay. To ensure the sufficient data coverage for high, medium and low copy transcripts, twelve indexed mRNA libraries were pooled and sequenced on an Illumina Nextseq 500 sequencer using TruSeq High Output Kit V2 150 cycles (FC-404-2001) in Paired-end E75 sequencing configuration. The raw data bcl files were de-multiplexed and converted into fastq files using Illumina bcl2fastq2 conversion V 2.19 software (illumina). We used FastQC to perform a quality control of the FASTQ files and STAR (GRCh38, Gencode25 and STAR 2.6.1b) to map the reads against the reference genome and count the number of reads uniquely mapping to each gene, for each sample. Heatmap plots of selected genes showing their variation among different samples were generated in R, version 3.5.1, using the heatmap 2 function of g plots library.

mRNA-Sequence and
Public domain of gene pathways (https://www.qiagen.com/us/) was used to retrieve genes related to apoptosis and DNA Damage repair. Gene Ontology Enrichment Analysis for differentially expressed genes was performed using the web-based tool Enrichr (6,7).